Methods: :
Rabbit LGACs were isolated according to established protocolsand cultured for 1-2 days prior to electroporation using theAmaxa (Lonza Group) nucleofector device and plasmid DNA (3 µg).The first plasmid used was Amaxa’s pmaxGFP control plasmid(3.4Kb). The second was a cathepsin-GFP fusion construct madeby using the pAcGFP1-N1 vector (Clontech) and mouse CathepsinS cDNA as template. The STOP codon was removed from the C-terminusby PCR and the sequence cloned in-frame into the vector. Thetransfected cells were seeded onto 6-well dishes or Matrigel-coated12-well plates and cultured for 24 or 48 hrs. After incubation,the cells were either processed for flow cytometry analysisor fixed and processed with appropriate primary and fluorescentsecondary antibodies and analyzed using confocal fluorescencemicroscopy.

Results: :
Flow cytometry analysis revealed that nucleofection of LGACswith a control GFP plasmid (pmaxGFP) could be optimized to resultin 47 ±2% transfection efficiency (n=4). The optimizedconditions included using 6 x 106 cells per nucleofection reactionon day 1 of cell culture, with 3 µg plasmid DNA, on programZ-001. Confocal fluorescence microscopy and labeling of actinfilaments and the mature secretory vesicle marker, Myosin 5crevealed that the normal morphology and secretory vesicle complementwas maintained in transfected reconstituted acini after electroporation.No significant morphological changes were observed in the reconstitutedacini, although additional single cells were detected in theculture. Electroporation on day 2 resulted in more reconstitutedacini than electroporation on day 1 although transfection efficiencywas slightly decreased. Additionally, transfection with a vesicle-targetedconstruct, cathepsin-GFP, resulted in overexpression of theprotein and punctate, vesicle-associated labeling in basolateralregions, consistent with targeting to lysosomes.

Conclusions: :
Nucleofection offers an efficient way to deliver plasmid DNAto previously difficult-to-transfect primary differentiatedepithelial cells. This technology offers a powerful, non-viraltool for further understanding of the lacrimal gland and relateddiseases.